Abstract Most antibiotics resulted from the Waxman platform, screening of soil microorganisms, but this limited resource was overmined by the late 60s. In the absence of a platform, compounds are introduced slower than pathogens acquire resistance, and the result is a human health crisis. The recent President's executive order ?Combating Antibiotic-Resistant Bacteria? underscores the significance of this problem. In this Program, we will develop an effective discovery program based on exploiting uncultured bacteria to resolve the bottleneck of antimicrobial drug discovery. Uncultured bacteria are an untapped source of secondary metabolites, and we developed methods to grow them and mine for antibiotic discovery. We discovered 25 new compounds from this source so far, including lassomycin, a novel compound with specific activity against the ClpP1P2C1 protease of M. tuberculosis; and teixobactin, a novel inhibitor of peptidoglycan synthesis which is essentially free of resistance development. However, the real potential of uncultured bacteria remains unrealized - the background of knowns and toxic compounds has been the main bottleneck even for this untapped source of chemical diversity. We propose to solve this problem by introducing transcriptome analysis as a rapid tool to identify promising compounds from uncultured bacteria. Compounds affecting the same target produce distinct transcription profiles that cluster together. This approach allows us to classify compounds as known; novel hitting a known target; novel hitting a new valuable target; hitting an undesirable target; or a nuisance compound lacking specificity. In a pilot study, we determined that crude extracts from producing strains can be used to generate transcriptome profiles in a test organism to identify targets, and deduce the presence of a potentially valuable compound. In the proposed project, we will create a database of transcription profiles from known antimicrobials, develop effective computational tools for transcriptome analysis, and will interrogate transcriptomes from a large number of extracts and their fractions from uncultured bacteria. Lead molecules will be validated in vitro and in an animal efficacy model. The end result of the project will be a novel discovery platform, new targets, and lead compounds for drug development. The project is a collaboration between Kim Lewis, PD/PI (NU), an expert in antimicrobial drug discovery and resistance; Karen Nelson, Co-Investigator (JCVI), an expert in genomics, meta-omics approaches and computational biology; and Amy Spoering, Co- Investigator (NovoBiotic), an expert in drug discovery from uncultured bacteria. These experts collaborated on producing preliminary data for this Program.